New sesquiterpenic acids from Inula wissmanniana

Fitoterapia 95 (2014) 139–146

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New sesquiterpenic acids from Inula wissmanniana Xiang-Rong Cheng a,b,1, Chun-Hui Wang a,1, Pan-Lei Wei a, Xu-Feng Zhang a, Qi Zeng a, Shi-Kai Yan a, Hui-Zi Jin a,⁎, Wei-Dong Zhang a,c,⁎⁎ a b c

School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, PR China School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China

a r t i c l e

i n f o

Article history: Received 3 January 2014 Accepted in revised form 11 March 2014 Available online 29 March 2014 Keywords: Inula wissmanniana Asteraceae Sesquiterpenic acids RAW264.7 macrophages NO production

a b s t r a c t Eight new (1−8) and two known (9 and 10) sesquiterpenic acids featuring α-methyleneγ-carboxyl units were isolated from the whole plants of Inula wissmanniana, along with two new germacranolides (11 and 12). Their structures were elucidated based on detailed spectroscopic analysis, including HRESIMS, 1D and 2D NMR, and X-ray crystallography. Notably, the skeleton of 1 was firstly discovered from nature, while that of 2 was discovered for the second time. All the compounds were evaluated for their inhibition against LPS-induced nitric oxide (NO) production in RAW264.7 macrophages. Compound 11 exhibited the strongest activity with the IC50 value of 1.04 μM. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The genus Inula (Asteraceae family) comprises 100 species distributed in Asia, Europe, and Africa, 20 species of which are found in China and mainly in the West and Southwest [1]. Previous reports have revealed high content of sesquiterpenoids in plants of the genus Inula, together with their remarkable biological activities, especially in anti-tumor and antiinflammation [2–8]. Inula wissmanniana is abundant but confined in Yunnan province, PR China, of which roots have been used as a traditional medicine for the treatment of indigestion in children [9]. Some eudesmanolides, germacranolides, thymols, inositols, and flavonolignans have been reported with potential anti-inflammatory activities [9,10]. With the purpose of replenishing previous researches on sesquiterpenoids from

⁎ Corresponding author. Tel./fax: +86 21 34205989. ⁎⁎ Correspondence to: W.-D. Zhang, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China. Tel./fax: +86 21 34205989. E-mail addresses: [email protected] (H.-Z. Jin), [email protected] (W.-D. Zhang). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.fitote.2014.03.013 0367-326X/© 2014 Elsevier B.V. All rights reserved.

I. wissmanniana, we report the isolation, structural elucidation, and nitric oxide (NO) inhibitory evaluation of eight new (1−8) and two known (9 and 10) sesquiterpenic acids, as well as two new germacranolides (11 and 12). 2. Experimental 2.1. General procedures Optical rotations were measured on a Perkin-Elmer 341 digital polarimeter. The IR spectra were obtained on a Bruker FTIR Vector 22 spectrometer with KBr pellets. 1H and 13C NMR spectra were measured on a Bruker DRX-400 spectrometer. ESI-MS were recorded on a Varian MAT-212 mass spectrometer. The TOF-ESI spectra were carried out on a Q-Tof micro YA019 mass spectrometer. The normal phase silica gel (100−200, 200−300 mesh, Yantai), MCI gel (CHP20P 75−150 μm, Mitsubishi Chemical Co.), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for column chromatography, and precoated silica HSGF254 (10−40 μm, Yantai) plates were used for TLC analysis. HPLC and preparative HPLC were performed with SHIMADZU LC 2010AHT, Agilent Technologies

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X.-R. Cheng et al. / Fitoterapia 95 (2014) 139–146

chromatography (4 × 40 cm; eluting with MeOH-H2O, 9:1, v/v) firstly, then fraction A5 (1.8 g) was subjected to Sephadex LH-20 (3 × 100 cm) with MeOH as eluent, and further purified by preparative RP-HPLC (ACN: H2O: HCOOH, 30: 70: 0.1, v/v) to yield compounds 1 (31.0 mg, tR 19.4 min) and 2 (3.0 mg, tR 26.3 min). Similarly, fractions A7 (0.8 g) and A9 (3.6 g) were purified by Sephadex LH-20 (3 × 100 cm), then further prepared by preparative RP-HPLC eluted with 0.1% formic acid in 30% acetonitrile to give 3 (17.0 mg, tR 23.7 min), 10 (14.5 mg, tR 29.6 min), and 5 (20.3 mg, tR 37.4 min). Fraction A10 (7.5 g) was also subjected to Sephadex LH-20 (3 × 100 cm) eluted with MeOH to give five subfractions which were further purified by preparative RP-HPLC (ACN: H2O:HCOOH, 30:70:0.1, v/v) to yield compounds 4 (5.8 mg, tR 26.9 min), 6 (31.2 mg, tR 31.2 min), 7 (20.5 mg, tR 21.0 min), 8 (4.1 mg, tR 33.6 min) and 9 (22.7 mg, tR 27.2 min), respectively. Moreover, the purities of all isolates (N95.0%) were analyzed by HPLC on an Agilent 1200 series pump equipped with a diode array detector.

1200 series and SHIMADZU LPD-20A with a C18 column. Optical density was measured using a multifunctional microplate reader Spectromax M5 (Molecular Devices, American) in NO inhibition assay. 2.2. Plant material The whole parts of I. wissmanniana were collected from Pingbian county, Yunnan Province, PR China, in August 2010, and identified by Prof. Han-Ming Zhang, Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, PR China. A voucher specimen (No. DJ 20100801) is deposited at School of Pharmacy, Shanghai Jiao Tong University, Shanghai, PR China. 2.3. Extraction and isolation The air-dried and powdered whole parts of I. wissmanniana (10.0 kg) were percolated with 95% EtOH at rt, and the extract (631.4 g) was further partitioned into petroleum ether (PE), CH2Cl2, EtOAc, and n-BuOH soluble fractions. The PE fraction (133.2 g) was chromatographed on a silica gel column (100−200 mesh, 1.5 kg, 10 × 100 cm) eluting with PE-EtOAc (100:1 to 10:1 v/v, each 4 L) to obtain 10 fractions (Fr. 1−Fr. 10) analyzed by TLC. Fr. 9 (2.5 g) was then subjected to MCI gel column chromatography (4 × 40 cm; eluting with MeOH-H2O, 9:1, v/v), and then the subfraction 3 (689.3 mg) was separated by preparative RP-HPLC (RP18, 210 nm, 70% MeOH) to give compounds 11 (43.2 mg, tR 26.8 min) and 12 (22.1 mg, tR 32.5 min). The EtOAc fraction (35.2 g) was subjected to a silica gel column (100−200 mesh, 400 g, 10 × 30 cm) with the gradient CH2Cl2-MeOH (100:1 to 5:1, v/v, each 1.0 L) as eluents and 13 fractions A1−A13 were obtained. All fractions were applied to MCI gel column

Table 1 1 H (400 MHz) and No.

1 2 3 4 5

1

7 8

δC

3.86 s

83.8 d 209.4 s 131.5 s 177.0 s 31.7 t

1.80 m 2.10 brd (13.2) 1.52 m

13 14 15

c

3b

δH

41.2 d 28.8 t 39.2 t

δC

6.94 d (7.7) 6.85 d (7.7)

2.97 2.68 2.90 2.11 1.68 2.83 2.72

46.7 s

10 11 12

b

2a

δH

9

2.3.2. 14(10 → 1),15(4 → 2)-Abeo-7αH-eudesm-1,3,5(10), 11(13)-tetraen-12-oic acid (2) White amorphous powder; [α]25 D + 38.2 (c 0.10, MeOH); UV (MeOH) λmax (logε): 220 (2.85) nm; IR (KBr) vmax 2936, 1693, 1625, 1441, 1382, 1328, 1276, 1162, 1029 cm−1; 1H

C (100 MHz) NMR data for compounds 1−4.

a

2.90 brd (13.3) 2.33 t (13.3) 2.59 m

6

a

13

2.3.1. (2S,7R,10R)-2-Hydroxy-1-nor-3-oxoeudesm-4,11(13)dien-12-oic acid (1) Colorless square crystals (MeOH); mp 102−105 °C; [α]25 D − 39.9 (c 0.10, MeOH); UV (MeOH) λmax (logε): 210 (2.65) nm; IR (KBr) νmax 3419, 2948, 2838, 1701, 1638, 1436, 1384, 1024 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 273.3 [M + Na]+, 249.3 [M − H]−; HRESIMS m/z 251.1284 [M + H]+ (calcd. for C14H19O4, 251.1283).

brd (15.2) m m m (overlap) m m m

c

~147 s ~171 sc 123.7 t

6.23 s 5.71 s 1.12 s

20.9 q

1.66 s

7.7 q

134.3 134.8 127.3 126.2 133.6

4a

δH s s d d s

δC

2.32 t (4.7) 2.51 brs

36.0 t

2.39 m

29.4 t

34.8 d 29.0 t

2.78 m 1.77 dd (15.0,7.5)

40.9 d 28.1 t

27.5 t

2.43 t (7.5)

42.2 t

133.8 s 144.2 s 171.6 s 6.37 5.69 2.14 2.26

s s s (overlap) s

Measured in CD3OD. Measured in CDCl3. Approximate data speculated from HMBC spectrum.

212.6 s 35.2 t 32.6 t 175.5 s 139.3 s

125.5 t 14.9 q 20.4 q

δH

δC

5.81 d (10.1) 6.66 d (10.1) 1.81 m 1.92 1.69 2.48 1.74 1.52 1.93 1.44

m(overlap) m m m m m (overlap) m

139.3 s 144.6 s 170.2 s 6.17 5.53 2.10 2.05

s s s s

126.3 t 29.9 q 17.7 q

208.3 s 126.1 d 153.5 d 68.8 s 49.5 d 28.0 t 40.5 d 27.8 t 34.6 t 45.2 s 126.1 s 170.6 s

6.19 5.65 1.24 1.34

s s s s

123.3 t 19.7 q 29.0 q

X.-R. Cheng et al. / Fitoterapia 95 (2014) 139–146 Table 2 1 H (400 MHz) and No.

1 2

13

C (100 MHz) NMR data for compounds 5−8.

5a

6b δH (J in Hz)

δC

δH (J in Hz)

δC

δH (J in Hz)

δC

5.79 d (10.4)

206.5 s 126.0 d

3.95 brs 5.52 d (10.0)

76.2 d 133.4 d

3.45 d (5.2) 5.83 dd (9.8, 5.2)

72.6 d 128.6 d

73.7 d 31.9 t

151.3 d 73.2 s 79.7 s 33.4 t

6.08 brs

129.1 d 145.4 s 43.7 d 29.3 t

6.09 d (9.8)

128.5 d 147.9 s 73.6 s 34.9 t

3.29 2.20 2.05 5.44

7 8

2.96 1.73 1.52 2.12 1.62

1.79 m

14 15 a b

8b

δC

6.41 d (10.4)

10 11 12 13

7b

δH (J in Hz)

3 4 5 6

9

141

6.18 5.65 1.26 1.42

m m m m m

35.4 d 27.2 t 31.3 t 50.5 s 147.4 s 171.0 s 123.7 t

s s s s

23.2 q 23.8 q

2.07 1.79 1.20 2.44 1.58 1.34 1.93 1.23

6.06 5.60 0.61 4.87 4.75

brd (10.9) brd (12.9) m (overlap) m brd (12.4) m brd (12.6) m (overlap)

s s s s s

38.3 d 26.7 t 36.9 t 39.6 s 146.3 s 168.3 s 122.1 t 10.7 q 110.4 t

1.82 1.55 2.95 1.65 1.43 2.34 1.08

5.94 5.45 0.73 5.07 4.97

brd (12.8) brd (12.8) m brd (11.4) m m m

34.2 d 26.9 t 29.1 t 38.5 s 146.1 s 171.3 s 120.0 t

s s s s s

20.3 q 112.3 t

m (overlap) m m brs

5.38 brs 3.26 1.92 1.39 1.80 1.29

6.05 5.50 0.83 1.70

m (overlap) m m m m

s s s s

122.9 130.8 142.5 123.7

d s s d

38.0 d 25.4 t 33.9 t 37.5 s 146.2 s 168.1 s 122.5 t 16.9 q 19.7 q

Measured in CD3OD. Measured in DMSO-d6.

263.2 [M − H]−; HRESIMS m/z 265.1448 [M + H]+ (calcd for C15H21O4, 265.1440).

and 13C NMR data, see Table 1; ESIMS m/z 253.2 [M + Na]+, m/z 229.2 [M − H]−; HRESIMS m/z 229.1227 [M − H]−− (calcd for C15H17O2, 229.1229).

2.3.4. 4β-Hydroxy-5α,7αH-1-oxoeudesma-2,11(13)-dien-12-oic acid (4) White amorphous powder; [α]20 D +12.7 (c 0.10, MeOH); UV (MeOH) λmax (logε): 210 (2.68) nm; IR (KBr) νmax 3400, 2945, 2327, 1712, 1680, 1646, 1454, 1384, 1106, 1029, 832 cm−1; 1H

2.3.3. 1,10-Dioxo-7αH-chromolaev-4,11(13)-dien-12-oic acid (3) White amorphous powder; [α]25 D +23.4 (c 0.10, MeOH); IR (KBr) vmax 2946, 2836, 1713, 1644, 1436, 1384, 1027 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 287.2 [M + Na]+, m/z

Table 3 1 H (400 MHz) and No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1' 2' 3' 4' 1''

13

C (100 MHz) NMR data for compounds 11 and 12, recorded in CDCl3. 11a

12a

δH (J in Hz)

δC

δH (J in Hz)

δC

1.96 1.24 1.81 2.18 1.30

m m m (overlap) m m

25.1 t

1.45 m (overlap)

24.6 t

19.9 t 33.2 t

1.66 m 2.27 m

18.5 t 35.8 t

2.93 3.84 2.63 2.22 1.82 4.88 1.87

d (9.8) dd (9.8, 9.8) m m m (overlap) dd (11.7, 3.7) m

6.32 5.77 1.02 1.51

d (3.4) d (3.4) d (6.7) s

6.12 q (7.2) 2.0 brd (7.2) 1.86 brs

59.6 66.9 81.2 42.6 28.9

s d d d t

78.0 d 30.5 d 138.0 s 169.1 s 120.6 t 17.6 q 18.6 q 167.6 s 127.8 s 138.7 d 15.9 q 20.6 q

5.15 d (10.4) 4.77 dd (10.4, 10.4) 2.0 m (overlap) 1.85 m 1.45 m (overlap) 4.81 dd (11.5, 2.7) 2.02 m (overlap) 2.73 q (7.6) 1.20 d (7.6) 0.91 d (6.7) 1.82 s

6.08 q (7.2) 1.96 brd (7.2) 1.85 brs

140.1 s 125.5 d 80.7 d 44.4 d 26.2 t 78.7 d 29.8 d 39.5 d 179.6 s 10.4 q 17.2 q 16.9 q 167.5 s 128.0 s 138.3 d 15.9 q 20.6 q

142

X.-R. Cheng et al. / Fitoterapia 95 (2014) 139–146

and 13C NMR data see Table 1; ESIMS m/z 287.1 [M + Na]+, m/z 263.2 [M − H]−− HRESIMS m/z 265.1424 [M + H]+ (calcd. for C15H21O4, 265.1434). 2.3.5. 4α,5α-Dihydroxy-7αH-1-oxoeudesma-2,11(13)-dien-12-oic acid (5) White amorphous powder; [α]20 D +3.1 (c 0.10, MeOH); UV (MeOH) λmax (logε): 205 (2.67) nm; IR (KBr) νmax 3423, 2946, 1684, 1646, 1628, 1616, 1383, 1108, 1023, 826 cm−1; 1H and 13 C NMR data see Table 2; ESIMS m/z 303.4 [M + Na]+; HRESIMS m/z 303.1201 [M + Na]+ (calcd. for C15H20O5Na, 303.1203). 2.3.6. 1β-Hydroxy-5α,7αH-eudesma-2,4(15),11(13)-trien-12-oic acid (6) White amorphous powder; [α]20 D + 8.4 (c 0.10, MeOH); UV (MeOH) λmax (logε): 205 (2.66) nm; IR (KBr) νmax 3400, 2945, 1690, 1620, 1376, 1105, 1020, 945, 887 cm−1; 1H and 13 C NMR data see Table 2; ESIMS m/z 271.2 [M + Na]+; HRESIMS m/z 247.1346 [M − H]−−(calcd. for C15H19O3, 247.1248). 2.3.7. 1α,5α-Dihydroxy-7αH-eudesma-2,4(15),11(13)-trien-12oic acid (7) White amorphous powder; [α]20 D +65.9 (c 0.10, MeOH); UV (MeOH) λmax (logε): 210 (2.83) nm; IR (KBr) νmax 3425, 2947, 1685, 1645, 1623, 1385, 1120, 1058 cm−1; 1H and 13C NMR data see Table 2; ESIMS m/z 286.8 [M + Na]+; HRESIMS m/z 263.1287 [M − H]−−(calcd. for C15H19O4, 263.1289). 2.3.8. 1β-Hydroxy-7αH-eudesma-3,5,11(13)-trien-12-oic acid (8) White amorphous powder; [α]20 D + 6.1 (c 0.10, MeOH); UV (MeOH) λmax (logε): 210 (2.75) nm; IR (KBr) νmax 3415, 2945, 1680, 1642, 1455, 1381, 1098, 1015 cm−1; 1H and 13C NMR data see Table 2; ESIMS m/z 271.0 [M + Na]+; HRESIMS m/z 247.1349 [M − H]−−(calcd. for C15H19O3, 247.1348). 2.3.9. (4R,5R,6S,7S,9S,10R)-9-Angeloyloxy-4,5-epoxygermacra11(13)-en-12,6-olide (11) Colorless square crystals (MeOH); mp 167−175 °C; [α]20 D + 61.6 (c 0.10, MeOH); UV (MeOH) λmax (logε): 214 (2.89) nm; IR (KBr) νmax 2965, 1777, 1712, 1646, 1459, 1384, 1233, 1142, 1083, 1011 cm−1; 1H and 13C NMR data see Table 3; ESIMS m/z 371.1 [M + Na]+; HRESIMS m/z 349.2010 [M + H]+ (calcd. for C20H29O5, 349.2015). 2.3.10. 4E-9β-Angeloyloxy-7α,10α,11αH-germacra-4,11(13)dien-12,6α-olide (12) White amorphous powder; [α]20 D + 135.8 (c 0.10, MeOH); UV (MeOH) λmax (logε): 219 (3.02) nm; IR (KBr) νmax 2932, 1777, 1713, 1645, 1542, 1456, 1384, 1232, 1162, 1035, 981 cm−1; 1H and 13C NMR data see Table 3; ESIMS m/z 357.3 [M + Na]+; HRESIMS m/z 357.2023 [M + Na]+ (calcd. for C20H30O4Na, 357.2036). 2.4. Crystallographic data of compounds 1 and 11 2.4.1. Crystallographic data of compound 1 C14H18O4, MeOH, M = 250.28, monoclinic, space group P2 (1), a = 7.8421 (16) Å, α = 90°; b = 6.1232 (12) Å, β = 90.41°; c = 12.892 (3) Å, γ = 90°; V = 619.0 (2) Å3, Z = 2,

ρcalcd = 1.343 mg/m3, crystal size 0.30 × 0.20 × 0.10 mm3. Cu Kα (λ = 1.54178 Å), F (000) = 268, T = 296 (2) K. The final R values were R1 = 0.0312, and wR2 = 0.0832, for 4497 observed reflections [I N 2σ (I)]. The absolute structure parameter was 0.04 (18). 2.4.2. Crystallographic data of compound 11 C20H28O5, MeOH, M = 348.42, orthorhombic, space group P2 (1) 2 (1) 2 (1), a = 12.053 (2) Å, α = 90°; b = 12.394 (3) Å, β = 90°; c = 12.903 (3) Å, γ = 90°; V = 1927.6 (7) Å3, Z = 4, ρcalcd = 1.201 mg/m3, crystal size 0.30 × 0.22 × 0.20 mm3. Cu Kα (λ = 1.54178 Å), F (000) = 752, T = 296 (2) K. The final R values were R1 = 0.0351, and wR2 = 0.0948, for 13710 observed reflections [I N 2σ (I)]. The absolute structure parameter was 0.08 (17). Crystallographic data for 1 and 11 have been deposited at the Cambridge Crystallographic Data Centre (deposition NO. CCDC 936934 and 936933). Copies of the data could be obtained free of charge on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44 1223 336033 or e-mail: [email protected]). 2.5. Assay for inhibitory effects against LPS-induced NO production RAW264.7 cell lines were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) in a humidified atmosphere of 5% CO2 in air at 37 °C, then cultivated in 96-well plates at a concentration of 1 × 105 cell/mL, 50 μL/well. After 1 h incubation, each compounds dissolve in dimethyl sulphoxide (DMSO; Merck) and 2 μL LPS (Sigma; 2 μg/mL) was added to the wells. After 22 h of incubation at 37 °C, the nitrite concentration in the culture supernatant was measured by the Griess reaction, optical density was measured at 540 nm using a multifunctional microplate reader. Aminoguanidine (purity ≥98.0%, Sigma-Aldrich) was used as a positive control and exhibited its inhibition with the IC50 value of 0.79 ± 0.05 μM. 3. Results and discussion The PE- and EtOAc-soluble extracts of I. wissmanniana were subjected to repeated column chromatography to afford ten new (1−8, 11, and 12) and two known (9 and 10) sesquiterpenoids (Fig. 1). The known compounds were identified as 1α-hydroxy5α,7αH-eudesma-2,4(15),11(13)-trien-12-oic acid (9) [11] and 1β-hydroxyilicic acid (10) [12] by interpreting spectral data and comparing with literature values. Compound 1 was isolated as colorless square crystals in the MeOH solution. Detailed analyses of its 1D NMR data indicated the molecular formula of C14H18O4, in accordance with the positive HRESIMS (m/z 251.1284, [M + H]+), corresponding to six degrees of unsaturation. Its IR absorptions at 3418, 1701, and 1638 cm−1 suggested the presence of hydroxyls, carbonyls, and olefinic bonds, respectively. The 1H, 13C, and DEPT NMR spectra (Table 1) exhibited 12 clear carbon resonances ascribed to four quaternary carbons (δC 209.4, 131.5, 177.0, 46.7; of which one was keto carbonyl and two was olefinic quaternary carbons), two methines (of which one was oxygenated), four methylenes (including an olefinic one), and two methyls, accounting for

X.-R. Cheng et al. / Fitoterapia 95 (2014) 139–146

143

Fig. 1. Structures of compounds 1−12.

C12H17O2. Moreover, the methyl singlets in 1H NMR spectrum (δH 1.12, 1.66) indicated their connections to quaternary carbons. All protons were assigned to their corresponding carbons by a HSQC experiment. The 1H−1H COSY and HMBC experiments were further applied to establish the planar structure of 1. Analysis of the 1H−1H COSY plot suggested a proton sequence of H2-5/H-6/H2-7/H2-8 (Fig. 2). The HMBC correlations from H2-5 to C-4 and C-9 and from H2-8 to C-9 and C-13 further cyclized the proton sequence by quaternary carbons C-4 and C-9. Additionally, the HMBC correlations from H-1 to C-2, C-4, and C-8, from H3-13 to C-1, C-4, C-8, and C-9, and from H3-14 to C-2, C-3, and C-4 established a cyclopentane ring and positioned the methyls, hydroxyl, and keto carbonyl groups (Fig. 2). The residual two quaternary carbons together with the olefinic methylene (δH/δC 5.71, 6.23/123.7) were presumed to be an α-methylene-γ-carboxyl unit connected to C-6, as evidenced by the HMBC correlations from H2-12 to C-6, C-10, and C-11. The relative configuration of 1 was determined by a NOESY experiment. Moreover, the unusual C14-skeleton was confirmed by an X-ray analysis using Cu Kα radiation (Fig. 3), and the absolute stereochemistry of 1 was assigned as 1S,6R,9R. Moreover, compound 1 represented an irregular type of sesquiterpene with 14 carbons from nature, although the skeleton of which had been once synthesized [13]. Compound 2 exhibited an [M − H]−−ion at m/z 229.1227 in the negative HRESIMS, corresponding to the molecular formula C15H18O2. Its 1H and 13C NMR spectra clearly revealed the

presence of an aromatic ring and a characteristic α-methyleneγ-carboxyl unit (Table 1). On the basis of the HSQC experiment which ascribed all protonated carbons, the 1H–1H COSY and HMBC experiments further established the planar structure of 2 in a rearranged eudesmane framework (Fig. 2). Analysis of the 1 H−1H COSY plot gave out proton sequences of H-3/H-4 and H2-6/H-7/H2-8/H2-9. The key HMBC correlations of H-3/C-1, C-2, C-5; H-4/C-2, C-6, C-10; H3-14/C-2, C-10; and H3-15/C-1, C-3 located methyls at C-1 and C-2, respectively. Moreover, the coupling constants between H-3 and H-4 (7.7 Hz) confirmed the presence of ortho-methyls at C-1 and C-2. Thus, the structure of 2 was established as 14(10 → 1),15(4 → 2)-abeo-7αH-eudesm1,3,5(10),11(13)-tetraen-12-oic acid. To the best of our knowledge, compound 2 is the second instance in this framework from nature, of which the first one was a stress compound isolated from Nicotiana rustica induced with tobacco mosaic virus [14]. From a biogenetic view, this kind of sesquiterpenoids was probably rearranged from eudesmane or eremophilane sesquiterpenoids (Scheme 1). It seems probable that the rearrangement of cyclohexadienol (i) in the presence of acid would give the eremophilane iii, which was similar to the rearrangement of santonin under acid catalysis [15]. Furthermore, eremophilane iii might be rearranged to produce intermediate iv with a spiro [4.5] ring [16]. Finally, the rearrangement of methylene at C-6 to C-1 could produce compound 2. The formation of compound 2 might provide a new insight into the structural transformation

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Fig. 2. Key 1H–1H COSY and HMBC (H → C) correlations of compounds 1, 2, 4, and 11.

of eudesmane type sesquiterpenoids. It is obvious that the further clarification of this sketchy biosynthetic pathway calls for much more biological and chemical studies. Compound 3 was obtained as a white amorphous powder with a molecular formula C15H20O4 deduced from HRESIMS (m/z 265.1448, [M + H]+), corresponding to six degrees of unsaturation. Detailed analyses of its 1D NMR data suggested the presence of three carbonyls, two pairs of olefinic bonds, and eight sp3-hybridized carbons (Table 1), accounting for five degrees of unsaturation and indicating a monocarbocyclic of 3. The 1H–1H COSY and HMBC experiments further established the planar structure of 3 which was in a chromolaevane framework. Previously, we have reported postia secoguaianolide as the first

chromolaevane sesquiterpenoid from Inula genus [5], and herein 3 is the second instance from this genus. Finally, the structure of 3 was established as 1,10-dioxo-7αH-chromolaev-4,11(13)dien-12-oic acid. Compound 4 was obtained as a white amorphous powder. Its formula was determined as C15H20O4 by HRESIMS (m/z 265.1424, [M + H]+), corresponding to six degrees of unsaturation. The observed two carbonyls and two pairs of double bonds in the 13C NMR and DEPT spectra accounted for four degrees of unsaturation, further indicating a bicyclic carbon skeleton in 4. Its planar structure was further established by detailed analysis of 1H–1H COSY and HMBC spectra, bearing a eudesmane framework (Fig. 2). The HMBC correlations of H-2/

Fig. 3. X-ray crystal structure of compounds 1 and 11 (ORTEP drawing).

X.-R. Cheng et al. / Fitoterapia 95 (2014) 139–146

145

Scheme 1. Plausible biogenetic pathway for compound 2.

C-4, C-10; H-3/C-5, C-15; and H3-14/C-1 located a carbonyl, a double bond, and a hydroxyl at C-1, C-2(3), and C-4, respectively. The NOESY correlations of H-5/H-7, H3-15 revealed their α orientation, while the lack of correlations H3-14/H-5, H-7 revealed a β-oriented methyl (Fig. 4). Hence, the structure of 4 was elucidated as 4β-hydroxy-5α,7αH-1oxoeudesma-2,11(13)-dien-12-oic acid. Compound 5 was a hydroxyl analog of 4, which concluded by detailed analysis of its MS and 1D NMR data (Table 2). The 1 H–1H COSY and HMBC experiments unambiguously revealed a hydroxyl at C-5. The relative configuration of 5 was further established by a NOESY experiment. The key NOESY correlations of H3-14/H3-15, H2-6 indicated the β-oriented methyls and α-oriented hydroxyls. Therefore, the structure of 5 was elucidated as 4α,5α-dihydroxy-7αH-1-oxoeudesma2,11(13)-dien-12-oic acid. Compound 6 shared the same planar structure with 1αhydroxy-5α,7αH-eudesma-2,4(15),11(13)-trien-12-oic acid (9) [11], supported by their similar 1H and 13C NMR data. The key NOESY correlations of H-5/H-1, H-7 together with the weak spin splitting of H-1 (brs) (Table 2) [11] established a β-oriented

hydroxyl at C-1. Thus, the structure of 6 was elucidated as 1β-hydroxy-5α,7αH-eudesma-2,4(15),11(13)-trien-12-oic acid. Detailed comparison of MS and 1D NMR data between 7 and 9 (Table 2) revealed that 7 was a hydroxyl derivative of 9 at C-5. This inference was further confirmed by the 1H–1H COSY and HMBC experiments for 7. Moreover, the NOESY correlation of H-1/H3-14 and the coupling constant of H-1/ H-2 (5.2 Hz) revealed their β orientation, while the correlation of H3-14/H-6a revealed an α-oriented hydroxyl at C-5. Thus, the structure of 7 was elucidated as 1α,5α-dihydroxy7αH-eudesma-2,4(15),11(13)-trien-12-oic acid. Compound 8 was also a eudesmane sesquiterpene with a characteristic α-methylene-γ-carboxyl unit, which was deduced from its MS and NMR data (Table 2). Its planar structure was established by an HMBC experiment. The HMBC correlations of H3-14/C-1, C-5 and H3-15/C-3, C-5 located a hydroxyl at C-1 and double bonds at C-3(4) and C-5(6). The lack of NOESY correlation of H-1/H3-14 revealed a β-oriented hydroxyl at C-1. Thus, the structure of 8 was elucidated as 1β-hydroxy-7αH-eudesma-3,5,11(13)-trien-12oic acid.

Fig. 4. Key NOESY correlations of compounds 4 and 11.

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Compound 11 was a white amorphous powder with a molecular formula of C20H28O5 based on its positive HRESIMS ion at m/z 349.2010 [M + H]+. The presence of an αmethylene-γ-lactone moiety [δH 6.32 (d, J = 3.4 Hz), 5.77 (d, J = 3.4 Hz); δC 138.0, 169.1, 120.6] [4−6] and an angeloyl group [δH 1.86, 2.00, 6.12; δC 167.6, 138.7, 127.8, 20.6, 15.9] [10] were deduced from the 1H and 13C NMR spectra (Table 3). In addition, the residual oxygen atom together with the carbon resonances at δC 59.6 (C-4) and 66.9 (C-5) revealed the presence of an epoxy ring. By detailed analysis of the 1H–1H COSY spectrum, the spin systems of H2-3/H2-2/H2-1/H-10/H-9/H2-8/H-7/H-6/H-5 and H-10/H3-14 were concluded (Fig. 2). The key HMBC correlations of H3-15/C-3, C-4, C-5; H-6/C-4, C-5, C-11; and H-9/C-1′, C-7, C-14 further established the planar structure of 11 and positioned the substituents (Fig. 2). The relative configuration of 11 was determined by a NOESY experiment. The strong NOESY correlations of H-7/H-5, H-9, H-10 revealed their α orientation, while those of H-6/H3-15 revealed their β orientation (Fig. 4). The coupling constants of H-5/H-6 (9.8 Hz) and H-6/H-7 (9.8 Hz) also confirmed the trans-relationships of H-5/H-6 and H-6/H-7. Furthermore, the absolute stereochemistry of 11 was determined unambiguously by X-ray crystallographic analysis using Cu kα radiation (Fig. 3). Thus, the structure of 11 is (4R,5R,6S,7S, 9S,10R)-9-angeloyloxy-4,5-epoxygermacra-11(13)-en-12,6olide. Compound 12 was also a germacraenolide with a molecular formula of C20H30O4 as deduced from its HRESIMS (m/z 357.2023, [M + Na]+) and NMR data (Table 3). The presence of an α-methyl-γ-lactone moiety and an angeloyl group was clearly observed in its 1H and 13C NMR spectra. The comparison of NMR data between 12 and 11 coupled with confirmations achieved by a HMBC experiment suggested the presence of an olefinic bond at C-4(5) of 2 (Table 3). The NOESY correlations of H-7/H-9, H-10, H-11; H-6/H3-13 were observed and suggested the α orientation of H-7, H-9, H-10, H-11 and the β orientation of H-6 and H3-13. The coupling constant of H-5/H-6 (10.4 Hz) coupled with the NOEY correlation of H-5/H-7 revealed a trans-double bond at C-4(5). Therefore, the structure of 12 was established as 4E-9β-angeloyloxy-7α,10α,11αH-germacra4,11(13)-dien-12,6α-olide. NO plays an important role in the inflammatory process, and the NO inhibitors were considered as potential anti-inflammatory agents [6]. Some eudesmanolides, germacranolides, thymols, inositols, and flavonolignans from I. wissmanniana have been reported with strong NO inhibitory activities [9,10]. In the present study, all isolates were evaluated for their inhibition against LPS-induced NO production in RAW264.7 macrophages. Compound 11 exhibited strong NO inhibition with the IC50 value of 1.04 ± 0.07 μM, while compounds 1−10 and 12 exhibited weak NO inhibition (IC50 N 50 μM). Acknowledgments This work was supported by the program NCET Foundation, NSFC (81230090, 81102778), the Global Research Network for

Medicinal Plants (GRNMP), the Shanghai Leading Academic Discipline Project (B906), the Key Laboratory of Drug Research for Special Environments, PLA, the Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), the Scientific Foundation of Shanghai China (10DZ1971700, 12401900501), the National Major Project of China (2011ZX09307-002-03, 2011ZX09102-006-02), the National Key Technology R&D Program of China (2012BAI29B06), the 12th Five-Year Plan for Science and Technology Development (2012BAD33B05), and the Fundamental Research Funds for the Central Universities (JUSRP1052).

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.03.013.

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